A. Immunosuppression Resulting from Loss of MAF Precursor Activity PA1 B. The Origin of .alpha.-N-acetylgalactosaminidase PA1 1. Precursor activity of vitamin D-binding protein (Gc protein) in patient plasma/serum for the macrophage activating factor. PA1 Lyso-Pc (1 .mu.g/ml)+mouse peritoneal cells PA1 {30 min incubation at 37.degree. C. }.fwdarw.{washed with PBS} PA1 {3 hr cultivation at 37.degree. C.}.fwdarw.macrophage activation assay. PA1 {assay of superoxide generation} PA1 2. Detection of .alpha.-N-acetylgalactosaminidase in blood stream of cancer and HIV-infected/AIDS patients. PA1 Patient plasma/sera (1 ml)+30% and 70% saturated ammonium sulfate 70% precipitate.fwdarw.dissolved in 50 mM citrate phosphate buffer (pH 6.0).fwdarw.dialyzed against the same buffer at 4.degree. C. for overnight. PA1 Reaction mixture: 100 .mu.l of the dialyzed sample+1.0 ml of 50 mM citrate phosphate buffer (pH 6.0) containing 5 .mu.moles of p-nitrophenyl N-acetyl-.alpha.-D-galactosaminide as substrate. PA1 Incubation time: 60 min, terminated by adding 200 .mu.l of 0.5M Na.sub.2 CO.sub.3. PA1 1. Precursor activity of serum Gc protein of cancer and HIV-infected/AIDS patients. PA1 2. Assay procedure for .alpha.-N-acetylgalactosaminidase.
Inflammation results in activation of macrophages. Cellular membrane damage and the inflammatory process result in the release of lysophospholipids. Administration into mice of small doses (5-20 .mu.g/mouse) of lysophosphatidylcholine (lyso-Pc) and other lysophospholipids induced a greatly enhanced phagocytic and superoxide generating capacity of macrophages (Ngwenya and Yamamoto, Proc. Soc. Exp. Biol. Med. 193:118, 1990; Yamamoto et al., Inf. Imm. 61:5388, 1993; Yamamoto et al., Inflammation. 18:311, 1994). This macrophage activation requires participation of B cells and T lymphocytes and a serum vitamin D binding protein (DBP; human DBP is known as group specific components or Gc). Activation of mouse peritoneal macrophages by lyso-Pc requires modification of the Gc protein by stepwise association with .beta.-galactosidase of lyso-Pc-treated B cells and sialidase of T cells, to generate the macrophage activating factor (MAF), a protein with N-acetylgalactosamine as the remaining sugar moiety (FIG. 1a) (Yamamoto et al., Proc. Natl. Acad. Sci. USA. 88:8539, 1991; Yamamoto et al., J. Immunol. 151:2794, 1993). Thus, Gc protein is a precursor for MAF. Incubation of Gc protein with immobilized .beta.-galactosidase and sialidase generates a remarkably high titered MAF (GcMAF) (Yamamoto et al., Proc. Natl. Acad. Sci. USA. 88:8539, 1991; Yamamoto et al., J. Immunol. 151:2794, 1993; Naraparaju and Yamamoto, Immunol. Lett. 43:143, 1994; U.S. Pat. No. 5,177,002). Administration of a minute amount (10 pg/mouse; 100 ng/human) of GcMAF resulted in a greatly enhanced phagocytic capacity of macrophages. When peripheral blood monocytes/macrophages of 175 cancer patients bearing various types of cancer were treated in vitro with 100 pg GcMAF/ml, monocytes/macrophages (phagocytes) of all cancer patients were activated for phagocytic and superoxide generating capacity. This observation indicates that patient phagocytes are capable of being activated. However, the MAF precursor activity of plasma Gc protein was severely reduced in approximately one third of the cancer patient population. Loss of the MAF precursor activity prevents generation of MAF. Therefore, macrophage activation cannot develop in certain cancer patients. Since macrophage activation is the first step in immune development cascade, such cancer patients become immunosuppressed. This may explain at least in party why cancer patients die with overwhelming infections. About one third of the patients had moderately reduced MAF precursor activities while the remaining one third of the cancer patients had MAF precursor activities similar to those of healthy humans. Lost or reduced precursor activity of Gc protein was found to be due to deglycosylation of plasma Gc protein by .alpha.-N-acetylgalactosaminidase detected in a cancer patient's blood stream. Deglycosylated Gc protein cannot be converted to MAF (FIG. 1b). The source of the .alpha.-N-acetylgalactosaminidase appeared to be cancerous cells. Radiation therapy of cancerous lesions decreased plasma .alpha.-N-acetylgalactosaminidase activity with concomitant increase of precursor activity. This implies that radiation therapy decreases the number of cancerous cells capable of secreting .alpha.-N-acetylgalactosaminidase. Thus, plasma .alpha.-N-acetylgalactosaminidase activity has an inverse correlation with the MAF precursor activity of Gc protein. Both .alpha.-N-acetylgalactosaminidase activity and MAF precursor activity of Gc protein in a patient's blood stream can serve as diagnostic and prognostic indices.
Similarly, when peripheral blood monocytes/macrophages of 65 HIV-infected/AIDS patients were treated in vitro with 100 pg GcMAF/ml, the monocytes/macrophages of all patients were activated for phagocytic and superoxide generating capacity. However, the MAF precursor activity of plasma Gc protein was severely reduced in about 1/10 of the HIV-infected patient population and approximately 25% of AIDS patients. These patients' plasma Gc protein is deglycosylated by .alpha.-N-acetylgalactosaminidase detected in HIV-infected patients. HIV-infected cells appeared to secrete .alpha.-N-acetylgalactosaminidase. Thus, .alpha.-N-acetylgalactosaminidase activity and MAF precursor activity of Gc protein in the patient's blood stream can serve as diagnostic and prognostic indices.
In my prior two U.S. Pat. Nos. 5,177,001 and 5,177,002, the entire disclosures of which are incorporated by reference herein, as are my above cited journal articles, is disclosed various macrophage activating factors, processes for preparing them as well as methods of inducing macrophage activation in a person in need of such activation.
Loss of the precursor activity was found to be due to deglycosylation of plasma Gc protein by .alpha.-N-acetylgalactosaminidase detected in the patient blood stream. The source of the enzyme appeared to be cancerous cells. Ehrlich ascites tumor cells contain a large amount of .beta.-N-acetylglucosaminidase and a very small amount of .alpha.-N-acetylgalactosaminidase (Yagi et al., Arch Biochem Biophys. 280:61, 1990).
My data has indicated that both .beta.-N-acetylglucosaminidase and .alpha.-N-acetylgalactosaminidase were detected in tumor tissue homogenates as represented by enzyme activities (about 41.5 and 32.1 nmole/mg/min, respectively) of a lung tumor tissue. Similar results were also observed with eleven different tumor tissues including 4 lung, 3 breast, 3 colon and 1 cervix tumors, though the .alpha.-N-acetylgalactosaminidase activity varied from 5.9 to 50.8 nmoles/mg/min. Radiation therapy of cancerous lesions decreased plasma .alpha.-N-acetylgalactosaminidase activity with concomitant increase of precursor activity. This implies that radiation therapy decreases the number of cancerous cells capable of secreting .alpha.-N-acetylgalactosaminidase.
Similarly HIV-infected patients carry .alpha.-N-acetylgalactosaminidase activity in their blood stream. HIV-envelope protein was found to contain .alpha.-N-acetylgalactosaminidase activity. HIV-infected cells can secrete this enzyme in to blood stream, resulting in deglycosylation of Gc protein. This would cause immunosuppression in HIV-infected/AIDS patients.
Thus, both .alpha.-N-acetylgalactosaminidase activity and MAF precursor activity of Gc protein in patient blood stream can serve as excellent diagnostic and prognostic indices.